Previously we have shown that dexamethasone (DEX) enhances the antitumor activity and ligand binding of the active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25-D₃), in the murine squamous cell carcinoma model SCC VII/SF. DEX also reduces the hypercalcemia toxicity of 1,25-D₃ treatment. However, the mechanism of the enhanced antitumor activity has not been defined. Here, we demonstrate that both cell cycle arrest and apoptosis were enhanced by DEX, effects that were inhibited by RU486. We also demonstrate that vitamin D receptor (VDR) protein levels were increased by the combination of 1,25-D₃ and DEX above the level observed with 1,25-D₃ treatment alone, whereas protein levels of the heterodimeric partner of VDR, retinoid X receptor, were lower for the combination than for 1,25-D₃ alone. Glucocorticoid receptor protein levels and ligand binding were increased by 1,25-D₃ but not by the combination. Treatment with the combination of 1,25-D₃ and DEX did not result in greater activation of a vitamin D response element-reporter than 1,25-D₃ alone or of a glucocorticoid response element-reporter than DEX alone. Nevertheless, the levels of phospho-Erk1/2 and phospho-Akt, signaling molecules that are modulated in 1,25-D₃-treated squamous cell carcinoma cells, were reduced by the combination of 1,25-D₃ and DEX more than by either agent alone. These trends were also observed in vivo. Our results suggest the involvement of the Erk and Akt signaling pathways in the antiproliferative effects of the combination of 1,25-D₃ and DEX and that phospho-Erk1/2 and phospho-Akt may be useful markers of response to this combination.